Characterization of a Novel Amino-Terminal Domain From A Copper Transporting P-Type ATPase Implicated in Human Genetic Disorders of Copper Metabolism Michael DiDonato A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto O Copyright by Michael DiDonato 1999 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Omwa ON K 1A ON4 OrtawaON KlAON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microformy vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be p~tedor otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. This rhesis is dedicated ro the mentor): of Micltele DiDonato and Domenico Beftini ABSTRACT Characterization of a novel copper transporting P-type ATPase implicated in human genetic disorders of copper metabolism Degree of Doctor of Philosophy, 1999. Michael DiDonato Graduate Department of Biochemistry, University of Toronto The -70 kDa copper binding domain fiom the Wilson disease copper transporting P- tvpe ATPase was expressed and purified as a fiision to giutathione-S-transferase (GST). Following purification, a detailed study of the metal binding characteristics of the domain, as well as the stmctural consequences of metal binding, was carried out. In order to determine the rnetal binding specificity of the domain, immobilized metal ion affinity (IMAC) and blotting analyses were performed. The results of these analyses indicate that the domain is able to bind a wide range of transition metals with varying affinities. The apparent order of affinity was found to be: Cu(I)/Cu(II)>Zn(Li)>Ni(II)>Co(II).Copper bound to the domain is in the +I oxidation state as assessed using the colorimetric copper(1) chelator bathocuproine disulfonic acid (BCS) and x-ray absorption near edge structure (XANES) spectroscopy. Neutron activation analysis (NAA) indicated a coppedprotein stoichiornetry of 6.5 copper atoms / mole of protein. In competitive "~nblotting experirnents non-radioactive copper (Cu(1) or Cu(I1)) was able to inhibit %nc binding to the domain in a sigmoidal marner, suggesting the prcsence of cooperativity. This sigmoidal inhibition pattern was not observed for any of the other metaIs tested. Detailed structural studies using circular dichroism (CD) spectroscopy indicate that significant changes in both secondary and tertiary structures take iii place in the domain upon copper binding. Furthemore, the structural changes which occur upon the addition of zinc are significantly different fiom those which occur with copper. X- ray absorption spectroscopy (XAS) indicates that the copper atoms are ligated by two sulfur atoms in a distorted linear arrangement. The XANES spectra did not change significantly with incremental addition of copper to the domain indicating that al1 six binding sites may be stnrcturally sirnilar. No amount of experimentation can prove me right; a single experiment can prove me wrong. -Albert Einstein Acknowledgments I would like to thank al1 the people who, in one way or another, have helped me in my graduate career over the past five years. First and foremost I would like to thank my supervisor Dr. Bibudhendra Sarkar for allowing me the freedom to pursue my interests in his lab and for al1 his help and guidance over the years. 1 would also like to thank al1 the members of my cornmittee, Dr. David Mademan, Dr. Robert Moms and Dr. Diane Cox for al1 their suggestions, help and encouragement. I could not have reached this point without the help and assistance of my Iab-mates both past and present. To Suree, thank you for always bcing there as a friend and for your constant Stream of suggestions and solutions to rny problerns, you have been a tnie role model. Many thanks to Tokameh who shared with me both the pain and exhilaration of research during Our tour of duty in the lab. you were missed over the last year. 1 would also like to thank Nira and Loretta for al1 their help and assistance over the years. To al1 the members of the Sarkar Lab (past and present) Suree, Tokameh, Mike, Negah, Lakshmi, Andrew, Damiano, Xuefeng and Jin-you, thank you for making the lab a great place to work. 1 wil1 miss those lab lunches and the crosswords. 1 must also take this opportunity to thank the person who has had the most influence on my life, my wife Grace DeSantis. She has helped in more ways than 1 can say and has becn the rock which has anchored me in the Storm. She has always been there with her support and bas never tired of hearing my depressing research stories. Her intelligence and perseverance has always been an inspiration to me. 1 would also like to thank al1 rny fnends for their help, support and friendship over the years. Thank you to Nana and Natalie first for teaching me molecular biology and for making the Pulleyblank lab a great place, but more importantly for your fi-iendship and support through the "rough" times in grad school. To Voula, thanks for al1 the QC on my data presentation (it really paid off in Edmonton!), for cirafting me into the BGSU and for enlightening me on the "purpose" of glycerïn soap. To Dina, thanks for al1 the spanakopita and dolmathakia and for always having interesting stones to tell me. To Randy, thanks for getting me involved with Aliquotes and giving me an excuse to put up a web site, it was great whils it lasted. To everyone else on the third floor who I've missed, thanks for making rny stay at Sick Kids interesting and fun, you will al1 be missed. Lastly I would like to thank my famiiy for always supporting my interests and encouraging me to strive for higher goals. I love you al1 very much. vii Table of Contents ABSTRACT ACKNO WLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS CHAPTER 1 Introduction 1. Metals in Biological Systems 1.3 Form and Function of Copper in Bmlogical Systems I -2.1 Types of copper 1 2.2 Important copper-containing enzymes in humans 1.3 Copper Homeostasis in Humans 1.3.1 Copper uptake and circulation 1.3 -2 Membrane transport of copper . 1.3.3 Intracellular copper trafficking 1.3.4 Excretionofcopper 1.4 Cation Transporting ATPases 1.4.1 Types of cation transporting ATPases 1.4.1.1 P-type ATPases 1.4.1.2 V-type and F-type ATPases 1.4.2 Meakes and Wilson Disease Copper Transporting ATPases 1.5 Human Genetic Disorders of Copper Metabolism 1 S.1 Menkes disease 1 -5.1.1 Genetics of Menkes disease 1 S.1.2 Clinical and Biochemical findings 1.5.1.3 Currenttreatments 1 .S. 1 -4 Animal models viii Table of Contents (cont'd) 1 -5.2 Wilson disease 1 -5.2.1 Genetics of Menkes disease 1 -5.2.2 Clinical and Biochemical findings 1-5.2.3 Current treatments 1 -5.2.4 Animal models 1.6 Project Rationale CHAPTER 2 Materials and Metbods 2.1 DNA Preparation 2.1.1 Ce11 lines 2.1 -2 Preparation of competent BL2 1(DE3) and DH5a Cd1 2.1.3 Transformation of competent BL2 l(DE3) and DHSa CelL 2.1.3.1 Preparation of glyceroI stocks 2.1.4 Purification of plasmid DNA 2.1.5 DNA agarose gel electrophoresis 2.1.6 Purification of DNA restriction fiagrnents from agarose gels 2.1 -7 DNA Sequencing 2.1.8 Restriction enzyme digests and ligation reactions 2.2 Construction of Expression Vectors 2.2.1 Construction of @EX-WCBD 2.2.2 Construction of pGEX-WCBD(P) 2.3 Expression and Purification of GST-WCBD and GST-WCBD(P) 2.3.1 Buffer and reagent preparation 2.3.2 Expression of GST-WCBD and GST-WCBD(P) 2.3.3 Purification of GST-WCBD and GST-WCBD(P) 2.3 -4 SDS-PAGE 2.3.5 Protein quantitation 2.3.6 N-Terminal Amino Acid Sequencing 2.3.7 Arnino Acid Analysis 2.4 Removal of GST Fom GST-WCBD and GST-WCBD(P) 2.4.1 Thrombin cleavage of GST-WCBQ Table of Contents (cont'd) 2.4.1.1 Optimization of thrombin cleavage conditions 2.4.2 PreScissionm cleavage of GST-WCBD(P) 2.5 Characterization of the Metal Binding Properties of WCBD 2.5.1 Immobilized Meta1 Ion Afinity Chromatography (MAC) 2.5.2 65~n0Blotting Assay 2.5 -2.1 Effect of pH on 6S~n(l~Blotting Assay 2.5.2.2 Effect of DTT on 65~n(~~Blotting Assay 2.5.3 Compehtion "z~(II)Blotting Assay 2.5.4 Stoichiometry of metal binding 2.5.5 Neutron Activation Analysis (NAAI 2.6 Characterization of the Copper Binding Sites 2.6.1 Preparation of apo-protein 2.6.2 Preparation of Samples For XAS and CD analysis 2.6.2.1 Preparation of Dry XAS Samples 2.6.2.2 Preparation of Liquid XAS and EPR Samples 2.6.3 EXAFS and XANES Analysis of Cu-GST-WCBD 2.6.4 Oxidative release of copper fiom GST-WCBD 2.7 Conformational Analysis of GST-WCBD and WCBD 2.7.1 Analysis of protein structural changes upon metal binding 2.7.2 Estimation of secondas.